PROJECT SUMMARY Malaria continues to be a scourge for humankind. The pervasiveness of disease, persistence of infection, and the complexity of the vector-borne transmission within populations together make disease elimination a huge challenge. With considerable effort, new drugs have been identified but they are few in number, and for malaria control and eradication efforts it is imperative to continue to prime the pipeline with new lead compounds. We have established the primate malaria parasite, Plasmodium knowlesi, as an in vitro model to study red blood cell invasion in our laboratory, and find that it possesses superior genetics and cell biology to the human malaria parasite, Plasmodium falciparum. We are now able to exploit P. knowlesi for forward genetic approaches with unprecedented efficiency. We will leverage the experimental tractability of the P. knowlesi parasite, which is amenable to continuous culture in vitro at the blood-stage, exhibits high transfection efficiency, and possesses a reduced AT-biased genome composition compared to other Plasmodium parasite species to facilitate the molecular cloning of large DNA fragments in fosmids. Here, we propose to establish a novel forward genetics approach (Fos-Seq) based on overexpression in P. knowlesi malaria parasites from fosmid DNA introduced through transfection, for the identification of genes that modulate the inhibition of parasites by specific small molecule inhibitors. Firstly, we will establish a robust selection protocol to identify fosmids containing genes known to modulate inhibition by antimalarial drugs. Secondly, we will then use this approach to interrogate the entire P. knowlesi genome by transfection with the fosmid library and inhibitor selection, followed by identification of candidate genes by next-generation sequencing. Finally, candidate genes will be validated through transgenic overexpression in parasites to assess their ability to modulate susceptibility to inhibitors. The application of Fos- Seq to parasite drug development, will provide a means for rapid, unbiased identification of genes associated with changes in susceptibility to specific inhibitors. We anticipate that this will aid in the identification of drug targets, cellular pathways that modulate drug activity, and resistance determinants that may arise in the field. More broadly, the approach could be used to deconvolute the genetic and molecular basis for many phenotypes identified in parasites.